The present invention relates to pyrrolobenzoxazepines, pyrrolobenzthiazepines and related compounds having the ability to induce apoptosis, to pharmaceutical compositions comprising these compounds and to their use as anti-tumour agents.
Benzodiazepines such as Valium are amongst the most highly prescribed drugs due to their anxiolytic, relaxant and sedative effects. Target-specific binding of benzodiazepines has been observed in many tissues and cell types and can be separated pharmacologically into two classes (see Table 1). The first and more widely studied of the two are the xe2x80x9ccentral-typexe2x80x9d binding sites located in brain which mediate the clinical effects of the benzodiazepines. These xe2x80x9ccentral-typexe2x80x9d sites are associated with the neuronal postsynaptic gamma-aminobutyric acid receptor which regulates Clxe2x88x92 flux (Costa and Guidotti, 1979). Benzodiazepines also bind to another distinct binding site (Braestrup and Squires, 1977; Schoemaker et al., 1981) found in the membranes of many mammalian tissues: heart, kidneys, lungs, adrenals, blood platelets and also the brain, termed the peripheral-type benzodiazepine receptor (PBR).
Apoptotic cell death can be induced by a variety of drugs with diverse chemical structure and different mechanism of action. Among the list of apoptosis inducing agents are a wide range of anti-cancer drugs including camptothecin, etoposide and the anthracycline antibiotics daunorubicin and doxorubicin. Although the mechanism of anti-tumour action of some of these drugs has not been fully elucidated, they ultimately activate the event of apoptosis in cells. All of the above anti-cancer drugs have previously been shown to induce apoptosis in cancerous cells derived from the haemopoietic system such as HL-60 and Jurkat cells. Any novel drugs that can also induce apoptosis in similar model cell lines may have potential as anti-cancer agents. One major problem in cancer chemotherapy is inherent or acquired resistance. Chronic myeloid leukaemia (CML) is associated with drug resistance, and although anti-cancer agents such as hydroxyurea appear to contain the disease during its chronic phase, progression to a fatal blast crisis is inevitable. Resistance to anti-cancer agents in CML has been suggested to be via suppression of apoptosis. It would therefore be useful to develop novel drugs that overcome this apoptosis suppression in CML cell lines.
The exact function of the PBR is unclear although PBR specific ligands such as Ro5-4864 (4xe2x80x2-chlorodiazepam) and PK11195 (1-(2-chlorophenyl)-1,3-dihydro-1-methyl-propyl)isoquinoline carboxamide) have been shown to elicit a wide variety of effects including alteration in cardiac action potentials and calcium channels, alterations of protooncogene expression modulation of steroidogenesis and alteration of immune function (Zisterer and Williams, 1997 for review). There have been many reports that benzodiazepines affect cell growth and differentiation in a number of cell types. These include induction of differentiation of Friend erythroleukemia cells (Wang et al., 1984a) and inhibition of cell proliferation (Wang et al., 1984b; Gorman et al., 1989; Ikezaki and Black, 1990; Camins et al., 1995). However, because of the high concentrations of PBR ligands necessary to elicit these antiproliferative effects and the demonstration of similar effects in cell lines which lack the PBR (Gorman et al., 1989), it could be concluded that these antiproliferative effects seem unrelated to a specific interaction of these drugs with this receptor.
Recently a novel series of high affinity PBR ligands based on a pyrrolobenzoxazepine skeleton, classified here as PBOX compounds, have been synthesised (Campiani et al., 1996). A recent study (Zisterer et al., 1998) has demonstrated that three of these novel PBR ligands, PBOX 1, 2 and 21 along with the classically used PBR ligands PK 11195 and Ro5-4864 were found to inhibit at micromolar concentrations and in a dose-dependent manner, the proliferation of rat C6 glioma and human 132 l N1 astrocytoma, without being cytotoxic. This antiproliferative effect was found to be mediated by arrest in the G1 phase of the cell cycle.
The present inventors have, while examining the effect of PK 11195, Ro5-4864 and some PBOX compounds on the proliferation of the human cancer cell lines, Jurkat (leukaemic T cell lymphoblast), HL-60 (promyelocytic leukaemia) HUT 78 (T cell leukemia), LAMA, KYO.1 and K562 cells which are all CML (chronic myeloid lymphoma) cells, Hela (cervix carcinoma), CEM (T lymphoblastoid) cells and MCF-7 (human breast carcinoma) cells, determined that these PBR ligands induce apoptosis, with various potencies, in the nine cell lines. Apoptosis is a cell suicide mechanism invoked in disparate situations, both physiological and pathological, to ablate unwanted, damaged, or potentially neoplastic cells. Apoptosis is classically defined by a characteristic set of morphological changes in the cell, including membrane blebbing, cell shrinkage, chromatin condensation, DNA fragmentation and eventual formation of membrane-bound apoptotic bodies. Although a number of reports have identified agents that are capable of inducing apoptosis in a variety of cells and tissues, the mechanisms responsible for the activation of the apoptosis process are poorly understood.